Induction of exon skipping in eukaryotic cells

Abstract

The present invention provides a method for at least in part decreasing the production of an aberrant protein in a cell, the cell comprising pre-mRNA comprising exons coding for the protein, by inducing so-call exon skipping in the cell. Exon-skipping results in mature mRNA that does not contain the skipped exon, which leads to an altered product of the exon codes for amino acids. Exon skipping is performed by providing a cell with an agent capable of specifically inhibiting an exon inclusion signal, for instance, an exon recognition sequence, of the exon. The exon inclusion signal can be interfered with by a nucleic acid comprising complementarity to a part of the exon. The nucleic acid, which is also herewith provided, can be used for the preparation of a medicament, for instance, for the treatment of an inherited disease.

1 . An isolated antisense oligonucleotide 17 to 40 nucleotides in length comprising a nucleotide sequence capable of binding to a purine-rich sequence of exon 51 of the human dystrophin pre-mRNA and inducing skipping of exon 51 of said pre-mRNA in a human muscle cell, wherein hAON#23: 5′-TGGCATTTCTAGTTTGG-3′ (SEQ ID NO: 18) binds to said purine-rich sequence and causes skipping of exon 51 of said pre-mRNA in a human muscle cell, said antisense oligonucleotide comprising a modification for increasing its resistance to an endonuclease in a cell. 
     
     
         2 . The oligonucleotide of  claim 1 , wherein the endonuclease is RNAse. 
     
     
         3 . The oligonucleotide of  claim 1 , which is a Peptide Nucleic Acid (PNA). 
     
     
         4 . The oligonucleotide of  claim 1 , wherein each T has been replaced by a U. 
     
     
         5 . The oligonucleotide of  claim 5 , wherein said oligonucleotide is a 2′-O-methyl oligoribonucleotide. 
     
     
         6 . The oligonucleotide of  claim 5 , which is a 2′-O-methyl phosphorothioate oligoribonucleotide. 
     
     
         7 . A nucleic acid delivery vehicle comprising a transcription unit expressing the antisense oligonucleotide of  claim 1 . 
     
     
         8 . A composition comprising the oligonucleotide of  claim 1 . 
     
     
         9 . A pharmaceutical formulation comprising the oligonucleotide of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         10 . A method for directing splicing of a dystrophin pre-mRNA in a cell by inducing the skipping of exon 51 of the dystrophin pre-mRNA to produce a dystrophin protein comprising:
 providing the cell with an antisense oligonucleotide of  claim 1  and wherein said oligonucleotide induces skipping of exon 51 from said dystrophin pre-mRNA in said cell.